Pursuant to 37 C.F.R. xc2xa71.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Genotype analysis has taken a more prominent position in the sphere of biological research, and particularly in the worlds of diagnostic and pharmaceutical research. In particular, the analysis of genotypes of individual members of the population is believed to be predictive of those individual""s predisposition to various diseases, as well as that individual""s responsiveness, sensitivity, etc. to therapies for diseases or other conditions. As such, genotype analysis is believed to have important application in both diagnostic and therapeutic applications. Of particular interest is the identification of different polymorphic variations in the genetic material of the members of a population, and the relationship of those polymorphic variations to disease predisposition or sensitivity to pharmaceutical therapy. For example, identifying which variant of a large number of different polymorphisms within a patient would be applied to determine the proper course of therapy for an individual, or to genetically counsel an individual to avoid high-risk behavior for a given disease for which that individual might be predisposed.
A wide variety of different technologies or different applications of similar technologies have been proposed and demonstrated for the identification of particular genetic variations as well as discrimination of those variations among members of the population. For example, arrays of oligonucleotide probes have been employed to effectively re-sequence portions of a target sequence known to contain a polymorphic locus. Such methods rely upon the differential hybridization of the target sequence to different probes on an array. While generally effective, such methods are very costly in terms of equipment and arrays, and are of relatively low throughput, permitting one or only a few experiments to be performed on an array at any given time.
U.S. Pat. No. 5,888,819 describes a method by which a probe that is hybridized to the polymorphic locus is extended by one base over the locus. The extension incorporates a base having a distinguishable label associated with it, allowing identification of the incorporated base, and thus identification of the variation.
A variety of other similar methods are available that use target specific probes at or near the variant locus, and give a detectable signal if and when perfect or imperfect hybridization occurs, e.g., through the exonuclease mediated activation of a FRET pair dye, e.g., through cleavage of one member of the pair from the probe. Such methods include TaqMan(copyright) and Invader(trademark) based technologies, available from Applied Biosystems, Inc. and Third Wave Technologies, Inc., respectively.
While all of the above methods are useful in polymorphism discrimination, such methods all suffer from problems of expensive reagents, equipment, or complex processes. Accordingly, it would be desirable to provide a discrimination method that is relatively simple, inexpensive and which does not require extremely expensive equipment, while still providing relatively high throughput and accuracy.
The present invention provides novel, simplified methods of identifying a nucleotide in a particular position of interest in an oligonucleotide sequence. The methods and systems are particularly useful in identifying polymorphic variants in genetic material, e.g., SNPs, STRs, deletions, insertions, and the like.
In a first aspect, the present invention provides methods of identifying a nucleotide in at least a first position in a polynucleotide sequence. In these methods, a polynucleotide target sequence is provided. The target sequence is hybridized with a first oligonucleotide probe, wherein the probe comprises a first subsequence of nucleotides, a first terminal nucleotide, and a first fluorescent label. The subsequence is complementary to a portion of the target sequence that is immediately adjacent to the first position, and the terminal nucleotide is complementary to one possible nucleotide in the first position. The hybridized probe and target sequence are then contacted with polymerase extension reagents in a first extension reaction mixture. By monitoring a fluorescent signal from the hybridizing and contacting steps one can identify the presence or absence of polymerase extension of the probe, the presence of polymerase extension of the probe indicating that the terminal nucleotide is complementary to the nucleotide in the first position. From that determination, one can identify the nucleotide in the first position.
The present invention also provides systems for carrying out these methods. Specifically, in at least one aspect of the invention there is provided a system for identifying at least a first oligonucleotide in a target nucleic acid sequence. The system comprises a reaction vessel having a first target nucleic acid sequence having an unknown nucleotide at a first position disposed in it. Also included is a first oligonucleotide probe having a first subsequence of nucleotides, the first subsequence being complementary to a subsequence of nucleotides in the target sequence that are immediately adjacent to the first position, a terminal nucleotide that is positioned to be adjacent to the first position when the first subsequence of the probe is hybridized to the subsequence of the target, and a fluorescent label. Further included are polymerase extension reagents. The system also includes a detector configured to monitor a fluorescent signal from the reaction vessel that is indicative of a presence or absence of polymerase extension of the probe.